Treatment of substrates for immobilizing biomolecules

ABSTRACT

A method of treating a substrate for immobilizing a biomolecule and substrates produced by the method are disclosed. The method includes contacting at least a portion of a substrate with a reducing agent such as a hydride. Treatment with an appropriate reducing agent substantially eliminates autofluorescence on substrates.

FIELD OF THE INVENTION

[0001] This invention relates to substrates for use in immobilizingbiomolecules and methods of making such substrates. More particularly,the present invention relates to treating substrates with a reducingagent and substrates produced by such treatment.

BACKGROUND OF THE INVENTION

[0002] Analysis of the structure, organization and sequence of nucleicacid molecules is important in the prediction, diagnosis and treatmentof human disease and in the study of gene discovery, expression anddevelopment. One laboratory tool used in the analysis of nucleic acidmolecules is the microarray or high density array (HDA), which is amicroarray containing a large number of targets per square centimeter ofarray surface. The microarray provides the framework for immobilizationof nucleic acid molecules for analysis on a rapid, large-scale basis.Microarrays generally include a substrate having a large number ofpositionally distinct nucleic acid targets attached to a surface of thesubstrate for subsequent hybridization to a nucleic acid target. The keyto efficiently immobilizing nucleic acid molecules is the surfacechemistry and the surface morphology of the microarrays substrate.

[0003] Microarrays have led to advances in biochemistry, chemistry andengineering that have enabled the development of a new gene expressionassay. This “chip-based” approach utilizes microscopic arrays of cDNAsprinted on glass substrates as high-density hybridization targets.Fluorescent target mixtures derived from total cellular messenger RNA(mRNA) hybridize to cognate elements on the array, allowing accuratemeasurement of the expression of the corresponding genes. A fundamentalrequirement for gene expression analysis using microarrays is asensitive and robust method for detecting the hybridized sample to thetarget DNA immobilized on the array. When DNA microarrays are used tomeasure the relative expression of MRNA between two samples (e.g.experimental and control), the targets representing the two samples areeach labeled with a different fluorescent dye, mixed and hybridized withthe microarray. The ratio of the two dyes, which reflects the level ofdifferential gene expression, is obtained by analyzing the array at thetwo different wavelengths. Therefore, to a large extent, the microarrayperformance depends on the optimal and accurate detection offluorescence emitted by the fluorophores conjugated to the targetmolecules.

[0004] An important element for successful microarray expressionanalysis is the quality of the substrate onto which hybridizationtargets are spotted. Poor quality slides result in low nucleic acidbinding efficiency, poor spot morphology and fluorescent background thatis often both relatively high and non-uniform.

[0005] The surface of a substrate used for microarrays also must containa suitable functional group for attaching target biomolecules such asDNA to the substrate surface. Target biomolecules such as DNA will notattach to a naked glass substrate. There are two general functionalitieson glass substrates for attaching DNA to the substrate surface. One is asurface including an aldehyde functionality, which is used to covalentlyattach amino-modified DNA onto the surface by reaction with freealdehyde groups using Schiff's base chemistry. Another different type offunctionalization of a substrate surface involves non-covalentattachment. Amine and lysine coated slides are two examples of manycoatings that provide for non-covalent attachment of biomolecules suchas DNA to the surface of a substrate. Another example of a coating thatprovides for noncovalent attachment of biomolecules is a silane coating,such as an amino propyl silane. One shortcoming of substrates includingan aldehyde functional group is that the substrates typically must berinsed with a reducing agent to reduce free aldehydes on the surface ofthe slide and prevent attachment of target biomolecules to locations onthe substrate surface that do not contain biomolecules.

[0006] The surfaces of both organic and inorganic substrates can bemodified by the deposition of a polymeric monolayer coating or film toconstruct biomolecular assemblies. In addition, surface modification canalso be used to promote adhesion and lubrication, modify the electricaland optical properties of the substrate surface, and createelectroactive films suitable for various optical and electronic sensorsand devices.

[0007] As noted above, compounds with amine functionality have foundextensive application in the preparation of surfaces for nucleic acidhybridization. Due to their ability to bond to a substrate with ahydroxide and their ability to bond to nucleic acids with an amine,silane compounds are useful as surface coatings that will effectivelyimmobilize nucleic acids. One example of a silane used for biologicalassay preparation is gamma amino propyl silane (GAPS), which may bedeposited by a variety of methods, including CVD, spin coating, spraycoating and dip coating.

[0008] Fluorescence detection sensitivity is severely compromised bybackground signals, which may originate from endogenous sampleconstituents/surface to which the target is immobilized or fromnonspecific hybridization of probes to the target. Generally, thenonspecific signals referred to as background, but not the intrinsicauto-fluorescence, can be eliminated by a high stringency wash of arraysafter hybridization. The intrinsic auto-fluorescence of the arraysobscures the sensitivity of gene expression analysis to a large extentby hindering the detectability of the low-level specific fluorescentsignals.

[0009] Attempts to diminish or eliminate auto-fluorescence by selectingfilters that reduce the transmission of emission relative to excitationwavelength or by selecting filters that absorb and emit at longerwavelengths are partially successful, but still have limitations.Although narrowing the fluorescence detection bandwidth increases theresolution, it also compromises the overall fluorescence intensitydetected.

[0010] While the present invention should not be limited by a particulartheory of operation, it is believed that fluorescence is caused by anaptly conjugated electronic system in an organic molecule. There aremultiple potential sources of auto-fluorescence. Auto-fluorescence couldbe due to trace impurities of fluorescent molecules that typicallycontain single or conjugated pi bonding. In addition, during storage orprinting, adsorption and oxidation of some biological or chemicalcontaminants, could result in the emission of fluorescence.

[0011] With so many possible causative agents for autofluorescence, itappears that there are at least three possible ways to circumvent thismajor problem. A first way is to make an array without any contaminants.However, this is very difficult to achieve. A second way to eliminateautofluorescence is to wash out contaminants after arraying. A drawbackof this approach is that this method may take long time and may end upeither losing DNA targets partially or incomplete washing off thecontaminants. A third way involves the use of relatively simple chemicalmeans. Applicants have discovered a relatively rapid, reproducible andeasily applicable method of substantially reducing autofluorescence onslides including a functional group for non-covalent attachment to abiomolecule.

[0012] Previous studies with reducing agents to eliminateauto-fluorescence from paraffin embedded tissue sections were found tosignificantly decrease deceptive false positive fluorescent signals.Hydrides are known to reduce the conjugated system in organic molecules.Sodium borohydride and sodium cyanoborohydride are mild reagents andhydride donors, which are known to reduce double bonds in conjugatedsystems. They both work well to reduce background signals in tissuesection for histochemistry and cytochemistry studies. See, e.g.,Schnell, S A, Staines, S A and Wessendorf, M W. J., Reduction oflipofuschin like auto-fluorescence in fluorescently labeled tissue,Histochemistry Cytochemistry, 1999. 47 (6), 719-30; Clancy, B andCauller, L. J. J., Reduction of background auto-fluorescence in brainsections following immersion in sodium borohydride, NeuroscienceMethods, 83, 1998. 97-102; Beisker, W Dolberate, F. and Gray J W, Animproved immunocytochemical procedure for high sensitivity detection ofincorporated bromodeoxyuridine, Cytometry 1987: 8: 235-9; and WillinghamM C. J. Histochemistry Cytochemistry, Alternative fixation processingmethod for preembedding ultrastructural immunocytochemistry ofcytoplasmic antigens, 1983: 31-791-889.

[0013] Taking into account the wide variety of factors contributing toautofluorescence, techniques for the elimination of autofluorescence inone biological system containing a specific type of organic moleculesand chemicals would not be expected work in a system employing differentorganic molecules and chemicals. Furthermore, methods used in theelimination of autofluorescence problems in bulk tissue samples wouldnot be expected to be useful in the elimination of autofluorescense onsubstrates used for microarrays. Other researchers have suggestedeliminating autofluorescense on slides containing non-covalentattachment functionality through curing by baking at high temperatures.See, e.g., Super Microarray Substrates Handbook, Telechem International,Inc.//arrayit.com, www.arrayit.com, 1999. However, applicantsexperiments have found that curing of the slides in an oven was largelyineffective in reducing autofluorescence.

[0014] It would be useful to provide an improved method of treatingsubstrates for immobilization and hybridization of biomolecules such asnucleic acids and oligonucleotides. The method should have the abilityto be performed in a reproducible manner. It would also be advantageousto provide a substrate that has uniform surface characteristics andexhibits low background noise or autofluorescence when the substrate isanalyzed using fluorescence scanning.

SUMMARY OF INVENTION

[0015] Accordingly, the present invention generally provides a method ofimmobilizing biomolecules on a surface of a substrate that providesreduced levels of autofluorescence on the substrate. The method includesproviding a substrate having a first surface including a functionalgroup for non-covalent attachment to a biomolecule and contacting atleast a portion of the first surface with a reducing agent. The methodfurther includes attaching a biomolecule to the functional group.According to one aspect of the invention, the reducing agent is selectedfrom the group consisting of hydrides. Applicants have surprisinglydiscovered that treatment with a reducing agent such as a hydridesignificantly diminishes autofluorescence on the surface of thesubstrate as well as on the locations deposited on the substrate. In apreferred aspect of the invention, the reducing agent includes aborohydride, and more preferably, sodium borohydride. According to amost preferred aspect of the invention, the sodium borohydride is in asolution at a concentration ranging from 0.01% to 1% weight per unitvolume. Other potential reducing agents that may be used in accordancewith the invention include sodium cyanoborohydride and copper sulfate.

[0016] Another aspect of the invention relates to a method of reducingautofluorescence on substrate containing an array of biomolecules.According to this aspect of the invention, the method includes providinga substrate having an array of target biomolecules non-covalentlyattached to at least a first surface of the substrate, treating at leasta portion of the first surface of the substrate with a reducing agent,and hybridizing complementary biomolecules to the target biomolecules.After hybridization, the substrate is scanned. Preferably, thecomplementary target biomolecules contain a fluorescent label, and thestep of scanning the substrate includes scanning the substrate for thefluorescent label.

[0017] According to a preferred aspect of the invention, the solutionfor treating the substrate includes a hydride, and more preferably, aborohydride. Most preferably, the step of treating the substrate with areducing agent includes contacting at least a portion of the firstsurface of the substrate with an aqueous solution containing between 0.1and 1% sodium borohydride by volume. Another aspect of the inventionrelates to having an array of biomolecules non-covalently attachedthereto produced by the methods described above.

[0018] According to one aspect of the invention, the immobilizedbiomolecules are nucleic acid molecules or oligonucleotides. In apreferred aspect of the invention, the substrate is a high density arrayor microarray.

[0019] The present invention provides a simplified and reproduciblemethod of providing substrates for immobilizing biomolecules.Experimentation has indicated that the substrates produced according tothe invention have good stability, reproducibility and exhibit lowbackground noise. Additional features and advantages of the inventionwill be set forth in the following description. It is to be understoodthat both the foregoing general description and the following detaileddescription are exemplary and are intended to provide furtherexplanation of the invention as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1A shows a representative Cy3 image of a slide before washingwith any solution under a PMT setting of 950 volts;

[0021]FIG. 1B show a representative Cy3 image of a slide washed withouta reducing agent under a PMT setting of 950 volts;

[0022]FIG. 1C shows a representative Cy3 image of a slide washed with areducing agent under a PMT setting of 950 volts;

[0023]FIG. 2 is a bar graph depicting the reduction in backgroundfluorescence produced by washing slides with a reducing agent;

[0024]FIG. 3A shows a representative Cy3 image of a slide before washingwith any solution under a PMT setting of 800 volts;

[0025]FIG. 3B shows a representative Cy3 image of a slide washed withouta reducing agent under a PMT setting of 800 volts;

[0026]FIG. 3C shows a representative Cy3 image of a slide washed with areducing agent under a PMT setting of 800 volts;

[0027]FIG. 4 is a bar graph comparing the Cy3 RFU readings for theuntreated slide, the slide washed without reducing agent and the slidetreated with sodium borohydride;

[0028]FIG. 5A shows a representative post-hybridization image of a slideprinted with washed without reducing agent;

[0029]FIG. 5B shows a representative post-hybridization image of a slidewashed and treated with sodium borohydride;

[0030]FIG. 6A is a graph of net Cy3 RFU versus net Cy5 RFU for the slidewithout sodium borohydride treatment;

[0031]FIG. 6B is a graph of net Cy3 RFU versus net Cy5 RFU for the slidewith sodium borohydride treatment;

[0032]FIG. 7A shows a representative Cy3 image of a slide washed withoutreducing agent;

[0033]FIG. 7B shows a representative Cy3 image of slide washed withreducing agent sodium borohydride for 10 minutes;

[0034]FIG. 7C shows a representative Cy3 image of slide washed withreducing agent sodium borohydride for 20 minutes;

[0035]FIG. 7D shows a representative Cy3 image of slide washed withreducing agent sodium borohydride for 30 minutes;

[0036]FIG. 8 is a graph comparing the spot and surface fluorescence ofeach of the samples in FIGS. 7A-7D;

[0037]FIG. 9A is a representative Cy3 image of a subgrid of an array ofoligonucleotides without washing;

[0038]FIG. 9B shows a representative Cy3 image of a subgrid of an arrayof oligonucleotides using wash procedures without sodium borohydride;

[0039]FIG. 9C shows a representative Cy3 image of a subgrid of an arrayof oligonucleotides using wash procedures with sodium borohydride;

[0040]FIG. 10A shows a representative Cy3 image of a slide beforetreatment not treatment obtained at a PMT setting of 950 volts;

[0041]FIG. 10B shows a representative Cy3 image of a slide washedaccording to procedures described above without sodium borohydrideobtained at a PMT setting of 950 volts;

[0042]FIG. 10C shows a representative Cy3 image of a slide washedaccording to procedures described above with sodium borohydride obtainedat a PMT setting of 950 volts;

[0043]FIG. 10D shows a representative Cy3 image of a slide washedaccording to procedures described above with sodium borohydride andafter arraying and hybridization obtained at a PMT setting of 950 volts;

[0044]FIGS. 11A is a graph comparing Cy3 RFU readings prior tohybridization for unwashed slides, slides washed without reducing agentand slides washed with reducing agent; and

[0045]FIG. 11B is a graph showing Cy3 reading on slides washed withoutreducing agent and slides washed with reducing agent afterhybridization.

DETAILED DESCRIPTION

[0046] Reference will now be made in detail to the present preferredembodiment of the invention. The invention provides a method of treatingsubstrates for immobilization of a biomolecule and substrates producedby the method having a biomolecule immobilized thereon. Applicants havesurprisingly discovered that autofluorescence on the slide surfaceincluding a functional group for non-covalent attachment to abiomolecule as well as on target biomolecules can be substantiallyreduced by treating the surface of the slide with a reducing agent asdescribed further below.

[0047] According to the present invention, biomolecules are immobilizedon a surface of a substrate having a first surface including afunctional group for non-covalent attachment to a biomolecule. When thesubstrate used for immobilizing biomolecules is a glass substrate, it ispreferred that hydroxy functional groups are present. Amine moieties arealso preferably present to provide interaction with DNA and otherbiomolecules. Particularly preferred substrates include a surface coatedwith an amino propyl silane, such as gamma amino propyl silane.

[0048] Suitable substrates for this invention are those having a surfacethat is accessible to solvents. The substrate itself may take any shapeincluding, but not limited to, rectangular, square, circular,cylindrical, conical, planar and spherical. The interior surface of abottle or tubing could be used as a substrate. The preferred substratehas a planar shape, and may be formed into a variety of microarrays,HDAs, microplates and laboratory dishes.

[0049] For optical or electrical areas of application, the substrate canbe transparent, impermeable or reflecting, as well as electricallyconducting, semiconducting or insulating. For biological applications,the substrate material may be either porous or nonporous and may beselected from either organic or inorganic materials.

[0050] Inorganic substrate materials can include metals, semiconductormaterials, glass and ceramic materials. Examples of metals that can beused as substrate materials are gold, platinum, nickel, palladium,aluminum, chromium, steel and gallium arsenide. Semiconductor materialsused for the substrate material can include silicon and germanium. Glassand ceramic materials used for the substrate material can includequartz, glass, porcelain, alkaline earth aluminoborosilicate glass andother mixed oxides. Further examples of inorganic substrate materialsinclude graphite, zinc selenide, mica, silica, lithium niobate, andinorganic single crystal materials.

[0051] Organic substrate materials are typically made from polymermaterials, due to their dimensional stability and resistance tosolvents. Examples of organic substrate materials are polyesters, suchas polyethylene terephthalate, and polybutylene terephthalate,polyvinylchloride, polyvinylidene fluoride, polytetrafluoroethylene,polycarbonate, polyamide, poly(meth)acrylate, polystyrene, polyethyleneor ethylene/vinyl acetate copolymer.

[0052] According to one embodiment of the invention, DNA oroligonucleotides are attached to a substrate having a coating or a layerincluding a functional group for non-covalent attachment to abiomolecule. Other biological or synthetic molecules can be attached tothe coated substrate. For example, other synthetic molecules include,but are not limited to, ribonucleic acids (RNA), deoxyribonucleic acids(DNA), synthetic oligonucleotides, antibodies, proteins, peptides,lectins, modified polysaccharides, synthetic composite macromolecules,functionalized nanostructures, synthetic polymers, modified/blockednucleotides/nucleosides, modified/blocked amino acids, fluorophores,chromophores, ligands, chelates, and haptens.

[0053] To facilitate non-covalent attachment of biomolecules, it isdesirable for the coating or layer on the substrate to include one ofvarious functional groups. These functional groups may include, but arenot limited to, primary amines, propyl hydrocarbon chain segments,silanol groups and siloxane bonds. Although the invention should not belimited to a particular theory of operation, generally, immobilizationof molecules at a substrate surface occurs in two steps: attraction ofthe molecules to the surface and binding of the molecules to thesurface. Some or all of the functional groups exposed on the surface ofthe silsesquioxane coating may contribute the attraction and binding ofbiomolecules or biomaterials, resulting in their immobilization on thesubstrate. For example, a protonated primary amine is positively chargedand may charge-attract and bind biomolecules. Propyl hydrocarbon chainsare hydrophobic, and their hydrophobic interaction with hydrophobicsegments of biomolecules may assist in binding them to the surface.Other interactions between biomolecules and coated substrates are, ofcourse, possible and the above discussion is not intended to beexhaustive or limiting of the mechanisms, which may play a role in theimmobilization of biomolecules on coated substrates in accordance withthis invention.

[0054] According to method aspects of the present invention, a substrateincluding a coating to promote non-covalent attachment of biomoleculesis provided and treated with a reducing agent. As discussed above,variability in spot size and high background levels can be problematicin biomolecule hybridization and scanning for hybridization. Variabilityin spot size and high background levels can arise from non- uniformitiesin a slide's coating which, in turn, can result in a working surfacewhose hydrophilic/hydrophobic properties are non-uniform.

[0055] Various techniques are known in the art for immobilizing DNA andoligonucleotides on surfaces, essentially any of which can be used inthe practice of the invention. A discussion of representativeimmobilization techniques used in the art can be found in U.S. Pat. No.5,919,626 and the references listed in that patent. Similarly,immobilization techniques are known for other biomolecules, such asspecific binding members. Along the same lines, techniques forimmobilization of molecules useful in tissue culture systems, e.g.,collagen, are also well-known in the art. It is understood that surfacesproduced in accordance with the present invention can be used toimmobilize a variety of biomolecules including, but not limited to DNAarrays, oligonucleotides, protein arrays, antibody arrays, peptidearrays and cell arrays.

EXAMPLES Example 1

[0056] Cleaning of Slides

[0057] Corning Microarray Technologies (CMT™) GAPS™ coated slides, whichare 25×75 mm glass slides coated with an amino-silane surface chemistrythat enables the even immobilization of DNA were first cleaned asfollows. Three different groups of slides were generally provided. Afirst set of slides was used as received. A second set of slides wascleaned as follows:

[0058] 1. Freshly made pre-hybridization solution was preparedcontaining: 2×SSC/0.05% SDS/0.2% BSA.

[0059] 2. 100 ml of pre-hybridization solution was added into a Coplinjar and the solution was warmed up to 42° C. in a water bath (for about20-30 min), and the slides were soaked (maximum of 4 slides/jar) for 10min, at 42° C.

[0060] 3. The slides were then transferred to Coplin jar filled with1×SSC at RT for 2 minutes.

[0061] 4. There slides were transferred to Coplin jar filled with0.2×SSC at RT for 2 min.

[0062] 5. Step 4 was repeated twice.

[0063] 6. Slides were dried by spinning at 2000 rpm, 2 min, 25° C.

[0064] A third set of slides was cleaned and treated with reducing agentas follows:

[0065] 1. Freshly made pre-hybridization solution was preparedcontaining: 2×SSC/0.05% SDS/0.2% BSA.

[0066] 2. 100 ml of pre-hybridization solution was added into a Coplinjar and the solution was warmed up to 42° C. in a water bath (for about20-30 min), and the slides were soaked (maximum of 4 slides/jar) for 10min, at 42° C.

[0067] 3. The slides were transferred to Coplin jar filled with2×SSC/0.05% SDS/0.25% NaBH₄ at 42° C. for 15 min.

[0068] 4. The slides were then transferred to Coplin jar filled with1×SSC at RT for 2 minutes.

[0069] 5. There slides were transferred to Coplin jar filled with0.2×SSC at RT for 2 min.

[0070] 6. Step 5 was repeated twice.

[0071] 7. Slides were dried by spinning at 2000 rpm, 2 min, 25° C.

[0072] Printing and Hybridization

[0073] Printing the DNA targets was performed on the CMT-GAPS slidesavailable from Corning, Inc. using conventional procedures. It will beunderstood that reduction in autofluorescence according to the presentinvention does not depend on the type of printing technique.Accordingly, either contact printing or ink-jetting technologies can beused to print rnicroarrays. Targets were prepared by labeling RNA withreverse transcriptase and Cy3 and/or Cy5.

[0074] In Examples in which hybridization was performed, each array washybridized with a solution consisting of 29% formamide, 2.25×SSC, 6%dextran sulfate, 0.17 μg/μL poly A, 0.10 μg/μL Cot 1 DNA, 0.2% BSA, anda given amount of labeled cDNA. For hybridization, 60 μL of thissolution was spotted onto the array and then spread over the entiresurface using a 24 mm×60 mm coverslip (Lifterslip, Erie ScientificCompany). The arrays were incubated overnight at 42° C.

[0075] In Examples in which hybridization was performed, slides werewashed after hybridization according to the following procedures:

[0076] 1. Slides were soaked in 2×SSC, 0.05% SDS at 42° C. and thecoverslip was removed. The slides were transferred to a coplin jarcontaining 2×SSC, 0.05% SDS at 42° C. for 5 min. This washing procedurewas repeated twice.

[0077] 2. Slides were transferred to a coplin jar containing 1×SSC at RTfor 5 min. This washing procedure was repeated twice.

[0078] 3. Slides were transferred to a coplin jar containing 0.2×SSC, atRT for 2 min. This washing procedure was repeated thrice.

[0079] 4. Slides were spin dried. Imaging

[0080] A GenePix 4000A (Axon Instruments) fluorescence scanner was usedto obtain the Cy3/Cy5 fluorescence images using a PMT setting of 750-950volts. All images were analyzed using GenePix Pro 3.0 analysis software(Axon Instruments).

Example 1

[0081] FIGS. 1A-1C show representative Cy3 images of a 1000 genesub-grid of a microarrays of 2000 cDNA human DNA clones printed on CMTGAPS™ slides before and after treatment with 0.25% sodium borohydride,under a PMT setting of 950 volts. Referring to FIG. 1A which is an imageof a slide before washing with any solution, both positive and negativespots are observed, indicating that the target quality is inconsistentsince the spot fluorescent intensity either higher or lower than thebackground intensity on the GAPS surface. Analysis of the images showedthat the normal prewash couldn't get rid of autofluorescence on bothspots and the surface. FIG. 1B shows an image of a slide washedaccording to the procedures described above without sodium borohydride.Analysis of the images in FIG. 2B showed that the prewash withoutreducing agent could not reduce autofluorescence on both spots and thesurface. FIG. 1C shows an image of a slide treated according to theprocedures above with sodium borohydride. As FIG. 1C shows, treatmentwith sodium borohydride significantly reduces auto-fluorescence of bothsurface and spots on Human 2K array. FIG. 2 shows that the slidestreated with sodium borohydride reduced the relative fluorescence unitreading from the slides was reduced from approximately 1500 to less than500 when compared with an untreated slide and a slide treated withoutreducing agent.

Example 2

[0082] FIGS. 3A-C show representative Cy3 images of a 1000 gene sub-gridof an array of microarrays of 6000 cDNA human DNA clones printed on aCMT GAPS™ slides before and after treatment with 0.25% sodiumborohydride, under a PMT setting of 800 volts. FIG. 3A is an image of anuntreated slide. FIG. 3B is an image of a slide washed without treatmentwith reducing agent. FIG. 3C is an image of a slide washed and treatedwith sodium borohydride. FIG. 4 is a bar graph comparing the Cy3 RFUreadings for the untreated slide, the slide washed without reducingagent and the slide treated with sodium borohydride. As shown in FIGS.3A-C and FIG. 4, treatment significantly reduces auto-fluorescence onthe microarray. Analysis of the image in FIG. 2B shows that even thougha conventional pre-wash can remove autofluorescence on target spotspartially, it can not remove autofluorescence on the surface at all.However, as can be seen from FIG. 2C, when treated with reducing agent,the autofluorescence from both the spots and the surface issubstantially eliminated.

Example 3

[0083] To test if reducing agent treatment affects the targethybridization, self-self hybridization of microarrays of 6000 cDNA humanDNA cancer clones printed on a CMT GAPS™ slides (treated and untreatedwith sodium borohydride) with 0.25 μg of total human brain RNA throughlinear amplification and reverse transcription labeling was alsoperformed. FIG. 5A is an image a slide washed without reducing agent,and FIG. 5B is an image of a slide washed and treated with sodiumborohydride according to the procedures described above. FIG. 6A is agraph of net Cy3 RFU versus net Cy5 RFU for the slide without sodiumborohydride treatment and FIG. 6B is a graph of net Cy3 RFU versus netCy5 RFU for the slide with sodium borohydride treatment. FIG. 6A showsmore genes have higher Cy3/Cy5 between 100-1000 on the untreated slidesdue to the Cy3 autofluorescence, when compared with the graph in FIG.6B. This suggests that without sodium borohydride treatment, the Cy3auto-fluorescence significantly affected the signal ratio in comparisonto slides treated with 0.25% sodium borohydride. This ultimately led toimprovement in gene expression profile.

Example 4

[0084] The time course of the reduction process has also been tested.Representative Cy3 images of a 1000 gene subgrid of microarrays of 4000cDNA human DNA cancer clones printed on a CMT GAPS™ slides before andafter treatment with 0.25% sodium borohydride, under a PMT setting of800 volts are shown in FIGS. 7A-7D. FIG. 7A is a representative Cy3image of a slide washed without reducing agent. FIG. 7B is arepresentative Cy3 image of slide washed with reducing agent accordingto procedures described above with sodium borohydride for 10 minutes.FIG. 7C is a representative Cy3 image of slide washed with reducingagent according to procedures described above with sodium borohydridefor 20 minutes. FIG. 7D is a representative Cy3 image of slide washedwith reducing agent according to procedures described above with sodiumborohydride for 30 minutes. FIG. 8 is a graph comparing the spot andsurface fluorescence of each of the samples. As shown in FIGS. 7A-7D andFIG. 8, when the reduction process with sodium borohydride was extendedfrom 10 minutes to 30 min, greater elimination of auto-fluorescence wasachieved.

Example 5

[0085] In addition to above cDNA arrays, oligonucleotide arrayperformance on CMT GAPS™ slides has also been evaluated. FIG. 9A is arepresentative Cy3 image of a subgrid of an array of oligonucleotideswithout washing. FIG. 9B is a representative Cy3 image of a subgrid ofan array of oligonucleotides using wash procedures without sodiumborohydride as described above. FIG. 9C is a representative Cy3 image ofa subgrid of an array of oligonucleotides using wash procedures withsodium borohydride as described above. The representative Cy3 images inFIGS. 9A-9C were obtained under a PMT setting of 800 volts. Once again,NaBH4 treatment significantly reduces oligonucleotide arrayauto-fluorescence, for both targets and GAPS surface itself.

[0086] Hybridization of liver and bacterial on oligonucleotide arraysprinted on CMT GAPS™ slides also showed improved signal/noise ratioafter treatment with sodium borohydride in comparison to the untreatedarrays (not shown). Image analysis revealed that highersignal-to-background ratio was obtained on the treated slides mostly dueto the reduced background intensity.

Example 6

[0087] Experiments were performed to determine the effect ofpre-treatment of CMT GAPS™ slides prior to printing of targets andhybridization. FIG. 10A shows a representative Cy3 image of a slidebefore treatment not treatment obtained at a PMT setting of 950 volts.FIG. 10B shows a representative Cy3 image of a slide washed according toprocedures described above without sodium borohydride obtained at a PMTsetting of 950 volts. FIG. 10C shows a representative Cy3 image of aslide washed according to procedures described above with sodiumborohydride obtained at a PMT setting of 950 volts. FIG. 10D shows arepresentative Cy3 image of a slide washed according to proceduresdescribed above with sodium borohydride and after arraying andhybridization obtained at a PMT setting of 950 volts. FIGS. 11A and 11Bare graphs respectively showing the Cy3 RFU readings prior tohybridization and after hybridization. The image analysis results inFIG. 11A indicate that more than 80% of the autofluorescence on the GAPSsurface was reduced after sodium borohydride treatment. FIG. 11B showsthat excellent array and hybridization image was achieved with the slidetreated with sodium borohydride. Even thought FIG. 11B shows that anequivalent amount of hybridization signal was obtained on both thesodium borohydride-treated and slides washed without sodium borohydride,higher signal-to-background ratio observed on sodium-borohydride-treatedslide due to the significant reduction of surface autofluorescence.

[0088] It will be apparent to those skilled in the art that variousmodifications and variations can be made to the present inventionwithout departing from the spirit or scope of the invention. Forexample, a variety of substrates containing various functional groupsfor noncovalent attachment to various biomolecules may be used inaccordance with the present invention. Thus, it is intended that thepresent invention cover modifications and variations of this inventionprovided they come within the scope of the appended claims and theirequivalents.

What is claimed is:
 1. A method of immobilizing biomolecules on asurface of a substrate comprising: providing a substrate having a firstsurface including a functional group for non-covalent attachment to abiomolecule; contacting at least a portion of the first surface with areducing agent; attaching a biomolecule to the functional group.
 2. Themethod of claim 1, wherein the reducing agent includes a hydride.
 3. Themethod of claim 1, wherein the reducing agent includes a borohydride. 4.The method of claim 3, wherein the borohydride includes sodiumborohydride.
 5. The method of claim 4, wherein the sodium borohydride isin a solution at a concentration ranging from 0.01% to 1% by volume. 6.A substrate made in accordance with the method of claim
 2. 7. Asubstrate made in accordance with the method of claim
 5. 8. A method ofreducing autofluorescence on substrate containing an array ofbiomolecules comprising: providing a substrate having an array of targetbiomolecules non-covalently attached to at least a first surface of thesubstrate; treating at least a portion of the first surface of thesubstrate with a reducing agent; hybridizing complementary probebiomolecules to the biomolecules; and scanning the substrate.
 9. Themethod of claim 8, wherein the complementary probe biomolecules arelabeled with a fluorescent label.
 10. The method of claim 9, wherein thestep of scanning the substrate includes scanning the substrate for thefluorescent label.
 11. The method of claim 10, wherein the reducingagent includes hydrogen.
 12. The method of claim 11, wherein thereducing agent includes a hydride.
 13. The method of claim 12, whereinthe reducing agent includes a borohydride.
 14. The method of claim 13,wherein the step of treating the substrate with a reducing agentincludes contacting at least a portion of the first surface of thesubstrate with an aqueous solution containing between 0.1 and 1% sodiumborohydride by volume.
 15. The method of 14 wherein the aqueous solutioncontains between 0.2% and 0.3% sodium borohydride by volume.
 16. Asubstrate having an array of biomolecules non-covalently attachedthereto produced by the method of claim
 8. 17. The substrate of claim16, wherein the biomolecules are nucleic acids or oligonucleotides. 18.The substrate of claim 17, wherein the substrate is contains highdensity array of nucleic acids or oligonucleotides.
 19. A method ofeliminating autofluorescensce from a substrate coated with a silanecomprising treating at least a portion of a first surface of the slidewith a reducing agent.
 20. The method of claim 19, wherein the silanecoating includes an amino-silane.
 21. The method of claim 20, whereinthe silane coating includes gamma-amino-propyl-silane
 22. The method ofclaim 20, wherein the reducing agent includes a hydride.
 23. The methodof claim 22, wherein the reducing agent includes sodium borohydride.